Several pathogenic spore-forming bacteria, including the causative agent of anthrax Bacillus anthracis, are inhaled or ingested as spores and resume growth and toxin production in the host after germination. The spore protein coat, which encapsulates the spore, is an important factor in the infectious cycle, because of its effective protective function and its regulatory role in the germination process by controlling access of germinants to receptors located in the inner membrane of the spore. The spore coat is a morphologically complex structure composed of approximately 60 different sporulation proteins that assemble around the nascent spore. In this proposal, we will use an integrated systems biology approach to map protein interaction networks and identify key regulatory hubs that nucleate spore coat assembly- which may serve as markers for spore detection and/or as targets for control of spore germination in applications against bioterrorism. In addition to its relevance to public health issues, we envision that our project will illustrate the value of systems biology approaches for investigating the assembly of complex structures. We propose to use high throughput protein localization screens to characterize the protein interaction networks that govern spore coat assembly in the model organism Bacillus subtilis. Specifically, we will use a library of fluorescent protein fusions to all of the coat proteins in B. subtilis, to define the spatiotemporal hierarchy of deposition of spore coat proteins around the spore, to identify which morphogenetic proteins are required for the recruitment of all spore coat proteins and learn the transcriptional and post-translational mechanisms underlying the regulation of coat assembly. The nature of the interactions established between pairs of coat proteins will be demonstrated using a large scale yeast two hybrid assay and various focused biochemical approaches. Finally, we will use computational tools to integrate the data gathered from these experiments and obtain a comprehensive representation of the spore coat protein interaction network that will serve as a template for the study of other spore-forming bacteria, particularly B. anthracis. PUBLIC HEALTH RELEVANCE Our research will derive new hypotheses and mechanisms for the resistance properties of Bacillus spores and the mechanisms that control spore germination. We anticipate that our results will have an impact on the design of therapeutic approaches to detect and eliminate pathogenic spore-forming bacteria.